A major goal of this proposal is to elucidate processes by which innate immune sensing mechanisms program B lineage cells to produce protective anti-West Nile virus (WNV) IgM and IgG antibodies. Humoral immunity to WNV is both necessary and sufficient to protect hosts from disease, however, the early events that coordinate the initial innate and subsequence adaptive antibody (Ab) responses to WNV are not well understood. We will elucidate how dendritic cells (DCs) and B cells respond to WNV and how rapid IgM and IgG anti-WNV neutralizing Abs develop. We hypothesize that initial innate programming of DC subsets dictate both the quality and the duration of protective Ab responses to WNV. To investigate this hypothesis we will conduct the following Specific Aims: Aim 1: We will compare the course of pathogenic WNV Texas (WN-TX) and attenuated WNV Madagascar (WN-MAD) strains early after in vivo infection. We propose that high and low pathogenic WNVs differ in how they infect, alter and program DCs. We will quantify changes early after infection in DC subset numbers, survival, cytokine production and infection. With Project 1, we will determine what innate immune factors regulate initial DC responses and susceptibility to WNV infection by comparing DC responses and infection in wildtype (WT) mice vs. C3 and IPS-1 KO mice Using mixed bone marrow (BM) chimeras, in which DCs have been ablated, we will determine how DC ablation or the absence in DCs of IPS-1, TLR-3 or other innate immune factors affects early events after WNV infection. Aim 2: To determine the molecular and cellular requirements for the development of rapid IgM neutralizing antibody responses to WNV. To determine the role of marginal zone (MZ) B cells in protective IgM immunity to WNV, we will assess IgM responses and susceptibility of MZ B cell-deficient CD22 KO and Notch2fl/fl conditional KO mice. DCs play a pivotal role in rapid IgM responses to encapsulated bacteria; using mixed BM chimeras, in which DCs have been ablated, we will determine how DC ablation or the absence in DCs of IPS-1, BAFF or other innate immune factors affects the development of neutralizing IgM Ab responses. Aim 3: We will investigate if MZ B cells are required for immune IgM to promote protective IgG immunity. Delivery of Ag to MZ-associated DCs can lead to a strong IgG antibody response. We will examine if delivery of WNV E protein to MZ DCs can induce IgM and IgG neutralizing Ab responses, and if so whether this form of Ag delivery can be used to induce protective, sterilizing immunity to WNV. These studies will lead to new insights into how best to design vaccines to WNV and other flaviviruses.